NGR-bearing human adenovirus type 5 infects cells in flotillin- or caveolin-mediated manner depending on the NGR insertion site.

Human adenoviruses represent attractive candidates for the design of cancer gene therapy vectors. Modification of adenovirus tropism by incorporating a targeting ligand into the adenovirus capsid proteins allows retargeting of adenovirus towards the cells of interest. Human adenovirus type 5 (HAdV-C5) bearing NGR containing peptide (CNGRCVSGCAGRC) inserted into the fiber (AdFNGR) or the hexon (AdHNGR) protein demonstrated an increased transduction of endothelial cells showing expression of aminopeptidase N, also known as CD13, and αvβ3 integrin both present on tumor vasculature, indicating that NGR-bearing adenoviruses could be used as tools for anti-angiogenic cancer therapy. Here we investigated how AdFNGR and AdHNGR infect cells lacking HAdV-C5 primary receptor, coxsackie and adenovirus receptor, and we showed that both AFNGR and AdHNGR enter cells by dynamin- and lipid raft-mediated endocytosis, while clathrin is not required for endocytosis of these viruses. We present evidence that productive infection of both AdFNGR and AdHNGR involves lipid rafts, with usage of flotillin-mediated cell entry for AdFNGR and limited role of caveolin in AdHNGR transduction efficiency. Lipid rafts play important role in angiogenesis and process of metastasis. Therefore, the ability of AdFNGR and AdHNGR to use lipid raft-dependent endocytosis, involving respectively flotillin- or caveolin-mediated pathway, could give them an advantage in targeting tumor cells lacking HAdV-C5 primary receptor.